During the course of ligand binding, the hemoglobin tetramer may assume ten ligation states which differ in the number and configuration of ligated subunits. Recent thermodynamic studies have shown that the ten ligation states fall into three groups, each characterized by a different value of the "cooperative free energy". These three energy levels arise from structural changes which occur in the tetramer as ligands are bound, and suggest that the hemoglobin tetramer may exist in at least three different structural forms. A major goal of the proposed work is to determine structures of the singly-ligated and one of the asymmetric doubly-ligated tetrameres. These species are particularly interesting, as they are distributed in a cooperative free energy level different from that for unligated or fully-ligated hemoglobin, suggesting they may correspond to a different structural form. The second focus of the proposed studies is to determine the structural changes which occur in the mutant hemoglobin Ypsilanti (beta 99 asp-tyr). Recent thermodynamic studies have shown that the site-specific modification in hemoglobin Ypsilanti produces unusual changes in the stability of the fully-ligated and unligated tetrameres, resulting in a dramatically altered cooperative free energy. The structure of the fully-ligated (carbon monoxy) form has been solved at low resolution, and the results show that fully-ligated hemoglobin Ypsilanti exhibits a quaternary structure which is distinctly different from either unligated or fully-ligated normal human hemoglobin. This has not been observed for any other forms of human hemoglobin. These studies will help tie together a wealth of related structural and functional information on hemoglobin, enabling a more comprehensive understanding of the molecular mechanism of cooperativity. In addition, this work will have broad implications for the ability to make meaningful structure-function correlations in other protein systems.